Pantothenate, also known as pantothenic acid or vitamin B5, is a member of the B complex of vitamins and is a nutritional requirement for mammals, including livestock and humans (e.g., from food sources, as a water soluble vitamin supplement or as a feed additive). In cells, pantothenate is used primarily for the biosynthesis of coenzyme A (CoA) and acyl carrier protein (ACP). These coenzymes function in the metabolism of acyl moieties which form thioesters with the sulfhydryl group of the 4′-phosphopantetheine portion of these molecules. These coenzymes are essential in all cells, participating in over 100 different intermediary reactions in cellular metabolism.
The conventional means of synthesizing pantothenate (in particular, the bioactive D isomer) is via chemical synthesis from bulk chemicals, a process which is hampered by excessive substrate cost as well as the requirement for optical resolution of racemic intermediates (e.g., resolution of DL-pantolactone to obtain D-pantolactone for chemical condensation with P-alanine). Accordingly, researchers have recently looked to bacterial or microbial systems that produce enzymes useful in pantothenate biosynthesis processes (as bacteria are themselves capable of synthesizing pantothenate). In particular, bioconversion processes have been evaluated as a means of favoring production of the D isomer of pantothenic acid, e.g., using microorganisms which selectively hydrolyze a DL-pantothenic acid ester to D-pantothenic acid; microorganisms which selectively decompose L-pantolactone resulting in D-pantolactone alone; and microorganisms which selectively hydrolyze DL-pantolactone to D-pantoic acid.
There is still, however, significant need for improved pantothenate production processes, in particular, for processes requiring reduced quantities of substrates and/or less expensive substrates. To this end, methods of direct microbial synthesis have recently been examined as a means of improving D-pantothenate production. In microbes, pantothenate biosynthetis is a multistep pathway resulting in condensation of pantoate (derived from α-ketoisovalerate) and β-alanine to form D-pantothenate. The isoleucine-valine (ilv) pathway biosynthetic enzymes, acetohydroxyacid synthetase (the ilvBN or alsS gene product), acetohydroxyacid isomeroreductase (the ilvC gene product) and dihydroxyacid dehydratase (the ilvD gene product) catalyze the conversion of pyruvate to α-ketoisovalerate. The reactions are further catalyzed by the pantothenate (pan) pathway biosynthetic enzymes ketopantoate hydroxymethyltransferase (the panB gene product), ketopantoate reductase (the panE gene product), aspartate-α-decarboxylase (the panD gene product) and pantothenate synthetase (the panC gene product).
The genes encoding the enzymes involved in the biosynthesis of pantothenic acid in Salmonella typhimurium and Escherichia coli have recently been identified and characterized (Frodyma and Downs (1998) J. Biol. Chem. 273:5572-5576 and Jackowski (1996) pp. 687-694, In Neidhardt et al (ed.) Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd ed. Am. Soc. Microbiol. Wash, D.C.). In E. coli, for example, the biosynthesis of pantothenic acid consists of four key steps. The first reaction is catalyzed by the panB gene product, ketopantoate hydroxymethyltransferase, and uses the L-valine intermediate α-ketoisovalerate to generate ketopantoate, which is subsequently reduced to pantoate by the panE gene product, ketopantoate reductase. The panD gene product, aspartate-α-decarboxylase, generates β-alanine from aspartate. The panC gene product, pantothenate synthetase, subsequently ligates β-alanine with pantoate to yield D-pantothenate.
The authors Dusch et al. described the identification of the Corynebacterium glutamicum panD gene and reported that expression of the C. glutamicum panD gene in E. coli yielded a strain producing pantothenate with a specific productivity of 140 ng of pantothenate per mg (dry weight) per hour. (Dusch et al. (1999) Appl. Environ. Microbiol. 65:1530-1539).
The authors Sahm and Eggeling have further identified the Corynebacterium glutamicum panB and panC genes and have described a genetically engineered strain of C. glutamicum which overexpresses the panBC genes (Sahm and Eggeling (1999) Appl. Environ. Microbiol. 65:1973-1979). The engineered strain produces pantothenate, however, it was necessary to overexpress the genes responsible for α-ketoisovalerate production in the host organism in order that pantothenic acid production could be detected. Moreover, without the addition of β-alanine, no substantial amounts of pantothenate accumulated with the strain constructed.
Likewise, a method of producing D-pantothenic acid has been described that takes advantage of a sodium salicylate resistant mutant strain of E. coli which produces D-pantothenic acid when cultured in the presence of β-alanine (U.S. Pat. No. 5,518,906). Generation of E. coli strains resistant to α-ketoisovaleric acid and/or α-ketobutyric acid, and/or α-aminobutyric acid, and/or β-hydroxyaspartic acid and/or O-methyl-threonine, in addition to salicylic acid, further increased pantothenic acid production. Moreover, transformation of a plasmid DNA carrying the panb, panC and panD genes into the salicylic acid resistant mutant strain resulted in increased pantothenate production, however, up to 20 g/L β-alanine or more was fed in the examples given. The panB-panC-panD genes are clustered on the E. coli chromosome.
Finally, a method of producing D-pantothenic acid has been described which utilizes a salicylic acid-resistant, α-ketoisovalerate-resistant, α-ketobutyrate-resistant, β-hydroxyaspartate-resistant, o-methylthreonine-resistent E. coli strain transformed with pantothenate biosynthesis gene-containing DNA fragments and/or branched amino acid biosynthesis gene-containing DNA fragments and cultured in the presence of β-alanine (U.S. Pat. No. 5,932,457).
Pantothenate production in bacteria results from the condensation of pantoate and β-alanine and involves the pantothenate biosynthetic enzymes ketopantoate hydroxymethyltransferase (the panB gene product), ketopantoate reductase (the panE gene product), aspartate-α-decarboxylase (the panD gene product) and pantothenate synthetase (the panC gene product). Although pantothenate is biologically active as a vitamin, it is further metabolized in all cells to Coenzyme A (CoA) which participates as an acyl group carrier in the tricarboxylic acid (TCA) cycle, fatty acid metabolism and numerous other reactions of intermediary metabolism. The initial (and possibly rate-controlling) step in the conversion of pantothenate to Coenzyme A (CoA) is phosphorylation of pantothenate by pantothenate kinase. A pantothenate kinase activity was first identified in Salmonella typhimurium by screening for temperature-sensitive mutants which synthesized CoA at permissive temperatures but excreted pantothenate at non-permissive temperatures. The mutations were mapped in the Salmonella chromosome and the genetic locus was designated coaA. The gene encodes the enzyme that catalyzes the first step in the biosynthesis of coenzyme A from pantothenate (Dunn and Snell (1979) J. Bacteriol. 140:805-808). Escherichia coli temperature sensitive mutants have also been isolated and characterized (Vallari and Rock (1987) J. Bacteriol. 169:5795-5800). These mutants (named coaA15(Ts)) are defective in the conversion of pantothenate to CoA and further exhibit a temperature-sensitive growth phenotype, indicating that pantothenate kinase activity is essential for growth. Moreover, it was noted that CoA inhibited pantothenate kinase activity to the same degree in the mutant as compared to the wild-type enzyme.
Feedback resistant E. coli mutants (named coaA16(Fr)) have also been isolated that posses a pantothenate kinase activity that is refractory to feedback inhibition by CoA (Vallari and Jackowski (1988) J. Bacteriol. 170:3961-3966). The mutation responsible for the reversion is, suprisingly, not genetically linked to the coaA gene by transduction. Additional data described therein support the view that the total cellular CoA content is controlled by both modulation of biosynthesis at the pantothenate kinase step and possibly by degradation of CoA to 4′-phosphopantetheine.
The wild-type E. coli coaA gene was cloned by functional complementation of E. coli temperature-sensitive mutants. The sequence of the wild-type gene was determined (Song and Jackowski (1992) J. Bacteriol. 174:6411-6417 and Flamm et al. (1988) Gene (Amst.) 74:555-558). Strains containing multiple copies of the coaA gene possessed 76-fold higher specific activity of pantothenate kinase, however, there was only a 2.7-fold increase in the steady state level of CoA (Song and Jackowski, supra). It has further been reported that the prokaryotic enzyme (encoded by coaA in E. coli and a variety of other microorganisms) is feedback inhibited by CoA both in vivo and in vitro with CoA being about five times more potent than acetyl-CoA in inhibiting the enzyme (Song and Jackowski, supra and Vallari et al., supra). Moreover, it has been reported that the panB gene product in E. coli is inhibited by CoA (Powers and Snell (1976) J. Biol. Chem. 251:3786-3793). These data further support the view that feedback inhibition of pantothenate kinase activity is a critical factor controlling intracellular CoA concentration.
Using standard search and alignment tools, coaA homologues have been identified in Hemophilus influenzae, Mycobacterium tuberculosis, Vibrio cholerae, Streptococcus pyogenes and Bacillus subtilis. By contrast, proteins with significant similarity could not be identified in eukaryotic cells including Saccharomyces cerevisiae or in mammalian expressed sequence tag (EST) databases. Using a genetic selection strategy, a cDNA encoding pantothenate kinase activity has recently been identified from Aspergillus nidulans (Calder et al. (1999) J. Biol. Chem. 274:2014-2020). The eukaryotic pantothenate kinase gene (panK) has distinct primary structure and unique regulatory properties that clearly distinguish it from its prokaryotic counterpart. A mammalian pantothenate kinase gene (mpanK1α) has also been isolated which encodes a protein having homology to the A. nidulans PanK protein and to the predicted gene product of GenBank™ Accession Number 927798 identified in the S. cerevisiae genome (Rock et al. (2000) J. Biol. Chem. 275:1377-1383).